Function Analysis Of Rice LRR Receptor-like Kinase Gene OsRK1 In Regulating Drought And Salt Stress Responses

Drought and salt stress seriously affect the growth and yield of rice.When the external environment changes,plants will perceive external environmental signals and transmit them to the interior of cells,thereby initiating gene expression to relieve the damage caused by external environmental changes.Receptor-like protein kinases are a large family of membrane receptor kinases whose main function is to receive and transmit external signals,activate different phosphorylation pathways,and ultimately regulate the transcription and expression of downstream genes,thereby affecting plant growth and environmental adaptability.The receptor-like protein kinase family in plants has a large number of members,diverse functions,and complex regulatory mechanisms.Currently,the molecular mechanisms by which these enzymes regulate responses to abiotic stresses such as high salt and drought are poorly understood.This study investigated the biological function of OsRK1.a receptor-like kinase gene in rice,and preliminarily explored its molecular mechanism involved in salt and drought tolerance.First of all,based on genevestigator gene chip database analysis,it was found that the expression of an LRR receptor-like kinase gene OsRK1 in rice was strongly induced by salt and drought stress,indicating that OsRK1 gene may be involved in regulating drought and salt stress responses in rice.Using qRT-PCR to detect the expression level of OsRK1 gene under high salt and drought conditions,it was found that,consistent with the database results,OsRK1 gene was indeed up-regulated by salt or drought treatments.In order to study the function of OsRK1 gene in regulating stress response,rice over-expression lines,mutant lines,and Arabidopsis over-expression lines of OsRK1 were constructed.These materials were used to analyze the functional role of OsRK1 gene in plant responses to environmental stresses.Under high salt or drought conditions,it was found that the seed germination rate of the rk1 mutant line decreased,and the growth of roots and shoots was significantly inhibited.Compared with wild type rice,the mutant showed a more sensitive phenotype to salt and drought treatment,while the over-expression lines showed a relatively insensitive phenotype.Through solution culture and soil culture methods,we further investigated the contribution of OsRK1 gene to plant responses to stresses.We found that over-expression lines can enhance tolerance to salt and drought environments,while mutant lines increase sensitivity to stresses.These experimental data demonstrated that OsRK1 gene plays an important role in plant responses to adverse environments.A series of physiological and biochemical comparative analyses were conducted on wild type,mutant lines,and over-expression lines,including in vitro water loss rate of leaves,stomatal opening,proline and soluble sugar content,active oxygen accumulation,expression of antioxidant enzyme genes and stress resistance genes,further demonstrating that OsRK1 gene plays a positive regulatory role in salt and drought tolerance of rice.In order to study the molecular mechanism of OsRK1 regulating plant stress tolerance,a rice cDNA library was screened using a yeast two hybrid system and a Whirly family protein OsWHY2 was obtained.The interaction between the two proteins was further verified through a one-to-one yeast two-hybrid experiment in vitro.It is speculated that OsRK1 may participate in the abiotic stress response process by phosphorylating OsWHY2 and promoting OsWHY2 to enter the nucleus to regulate the expression of related genes.In order to search for downstream genes regulated by OsWHY2,this study carried out the transcriptome sequencing analysis on the drought treated rk1 mutant line using wild type rice as a control.The results showed that in the transcripts of | Log2(rk1/ZH11)|>1.0(P<0.05),1303 genes were up-regulated and 374 genes were down-regulated.According to KEGG analysis,it was found that most of the differential genes were concentrated in the phenylpropanoid metabolic pathway.Consistent with this,up-regulation of the key genes for phenylpropanoid metabolism,OsPAL4,OsPAL6,and OsPAL7,was found in the transcriptome.The expression changes of these PAL genes under high salt and drought conditions were validated by qRT-PCR method,and it was found that these PAL genes were significantly down-regulated after stress induction.Further,using OsRK1 over-expression lines and mutant lines,the possible effects of OsRK1 gene on PAL gene expression were analyzed.The results showed that OsRK1 gene can negatively regulate the expression levels of OsPAL4,OsPAL6,and OsPAL7 genes.These results suggest that the PAL gene may be a class of downstream genes regulated by the OsRK1-OsWHY2 interaction module,and also reflect the importance of phenylpropanoid metabolism in the OsRK1 pathway.In summary,this thesis has discovered a new receptor like kinase gene OsRK1 involved in drought and salt stress responses in rice,demonstrating its important function in positively regulating salt tolerance and drought tolerance in plants.At the same time,through the study of the mechanism of action of OsRK1,the importance of"OsRK1-OsWHY2-OsPALs" pathway was proposed in the action of OsRK1.This study not only provides a new perspective for understanding the regulatory mechanism of receptor-like kinase involved in salt and drought tolerance in plants,but also provides important stress tolerance gene resources for agricultural breeding,with potential application value.